Regulated Secretion of Acid Sphingomyelinase

Jenkins, Russell W.; Canals, Daniel; Idkowiak‐Baldys, Jolanta; Simbari, Fabio; Roddy, Patrick; Perry, David; Kitatani, Kazuyuki; Luberto, Chiara; Hannun, Yusuf A.

doi:10.1074/jbc.m110.125609

Cited by 94 publications

(65 citation statements)

References 62 publications

(52 reference statements)

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“…VSVG3-GFP was kindly provided by Dr. Chiara Luberto (Medical University of South Carolina), and was generated and described by the laboratory of Dr. Kai Simons (14). pDsRed-N1-MonomeraSMase WT was previously described (15). DsRed-aSMase S508A mutant was generated via site-directed mutagenesis using the QuikChange kit from Stratagene, as previously described (11).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation-Immunoprecipitation of V5-tagged proteins was performed as previously described (15). Briefly, V5-S-SMase was immunoprecipitated directly from conditioned medium with 1 g/ml of anti-V5 mAb, unless otherwise indicated, and 1:20 protein A/G-agarose beads (Santa Cruz) in the presence of 1:500 protease inhibitor mixture (Sigma) by rocking overnight at 4°C.…”

Section: R600hmentioning

confidence: 99%

“…SDS-PAGE and Immunoblotting-SDS-PAGE and immunoblotting were performed as described previously (15). Primary antibody dilutions were V5, 1:5,000; aSMase, 1:500 -1,000; DsRed, 1:500; and ␤-actin, 1:10,000.…”

Section: R600hmentioning

confidence: 99%

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A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Jenkins

Idkowiak‐Baldys

Simbari

et al. 2011

Journal of Biological Chemistry

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Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn 2؉ )-independent lysosomal aSMase (L-SMase) and a Zn 2؉ -dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn 2؉ -activated proenzyme to a mature 65 kDa, Zn 2؉ -independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn 2؉ -dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M r as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ⌬R608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.

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Section: Methodsmentioning

confidence: 99%

Section: R600hmentioning

confidence: 99%

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A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Jenkins

Idkowiak‐Baldys

Simbari

et al. 2011

Journal of Biological Chemistry

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“…In Vitro SMase Activity Assays-The acid SMase (aSMase) activity assay was performed essentially as described previously (41). Cells were harvested by centrifugation following a wash with cold PBS.…”

Section: Sphingolipid Analysis By Quantitative High Performance Liquimentioning

confidence: 99%

Ceramide Synthase-dependent Ceramide Generation and Programmed Cell Death

Mullen¹,

Jenkins

Clarke

et al. 2011

Journal of Biological Chemistry

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The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.

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“…In previous studies, factors of inflammation, including TNF-α, ROS, LPS, amyloid-β, IL-1β, and LDL, stimulated SMase activation activity, which then produced ceramide and initiated ceramide signal pathways to transfer death signaling [25][26][27]. This study evaluated the effects of LMG on bacterial SMase activity in vitro.…”

Section: Lmg Inhibited Smase Activitymentioning

confidence: 99%

β-Glucan Contents and Anti-wrinkling Effects of Brown Rice <i>Phellinus linteus</i> Mycelium Extracts Fermented with Lactobacillus plantarum

Kim¹,

Ahn²,

Kwon³

et al. 2016

Asian J Beauty Cosmetol

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The major cell wall constituent of Ganoderma lucidum (G. lucidum) is β-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weight β-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies of G. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H 2 O 2 -induced cell death in RAW264.7 cells, identifying that LMG protected cells from H 2 O 2 -induced damage. LMG treatment decreased H 2 O 2 -induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMases in vitro showed that LMG suppressed the activities of both neutral and acid SMases in a concentrationdependent manner. These results suggest that LMG, a water-soluble β-1,3-glucan recycled from extracted residue of G. lucidum, possesses antioxidant capability against H 2 O 2 -induced cell death by attenuating intracellular ROS and inhibiting SMase activity.

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Regulated Secretion of Acid Sphingomyelinase

Cited by 94 publications

References 62 publications

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Ceramide Synthase-dependent Ceramide Generation and Programmed Cell Death

β-Glucan Contents and Anti-wrinkling Effects of Brown Rice <i>Phellinus linteus</i> Mycelium Extracts Fermented with Lactobacillus plantarum

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